胎盘钙化组织中纳米细菌的分离培养与鉴定及其相关蛋白的初步分析

发布时间：2018-06-10 07:35

本文选题：纳米细菌 + 胎盘钙化　；参考：《重庆医科大学》2012年博士论文

【摘要】：背景：芬兰科学家Kajander教授及其团队最早从哺乳动物细胞培养过程发现细胞的空泡样变化，在排除了一切可能的微生物污染的情况下对其进行分析，发现了一种可以自我复制矿化且直径只有50-800nm的微生物，命名为纳米细菌（Nanobacteria NB）并申请了专利1。利用傅里叶变换色散光谱分析（FTIR）表明纳米细菌外壳的主要成分为羟磷灰石，所以又称为纳米矿化颗粒（CNPs）。有科学家在透射电镜下观察早期钙化胎盘组织发现纳米矿化颗粒的存在，但未能分离培养出纳米细菌。目的：从胎盘钙化组织和相应胎儿脐带血中分离培养出与钙化相关的纳米细菌，观察其生长特性，探讨胎盘钙化发生的微生物感染证据以及胎盘钙化与胎儿纳米细菌感染的关系。方法：以临床所取36份胎盘钙化组织作为研究对象，透射电镜观察钙化组织中的纳米细菌。而后依次利用1mol/L盐酸脱矿，Tris中和，生理盐水稀释，14,000g高速离心，最后通过0.22μm细菌滤器过滤的方法进行分离。滤液过滤到有棉塞试管中，加入含有10%胎牛血清的RPMI1640细胞培养基，调整pH7.4，放入37℃细胞培养箱,5％CO2，95％空气条件下培养。观察细菌生长情况并依据沉淀类型分类。OD650波长下记录纳米细菌菌液相对浓度OD值，制作生长曲线。胎牛血清（FBS）和生理盐水作为试剂对照,无钙化的正常胎盘组织作为实验对照，用RPMI1640培养基作为空白对照。同时对存在胎盘钙化的胎儿取脐带血，对纳米细菌进行分离培养，方法同上。结果：透射电镜下观察钙化胎盘组织标本，可见胎盘绒毛组织和钙化斑块之间存在椭圆形纳米颗粒，其直径约为50-500nm，每个颗粒都被具有不同电子密度的薄外壳包围，其中有些颗粒可见分裂，与国内外文献报道相似。实验组中有28例可见白色沉淀完全或部分粘附在试管底部，对照组未见沉淀产生，差异具有显著性（P 0.01）。OD650分光光度计下记录得到纳米颗粒生长曲线与其他细菌类似。脐带血分离培养的结果与相应钙化组织胎盘中纳米细菌培养的结果正相关,有12例实验组培养物可见白色沉淀物，对照组未见（P 0.01）。结论：通过纳米细菌分离培养的方法可以从钙化胎盘组织中得到具有自我复制矿化功能的纳米颗粒，且存在胎盘钙化的胎儿也较易感染此纳米颗粒。. 背景：进一步对培养所得白色沉淀物进行形态学和基因组学的鉴定。由于纳米细菌形态微小，有的还不足100nm，因此许多科学家认为它并不存在遗传基因，也不属于生物范畴。Kajander教授向GenBank中提交了纳米细菌特异的16SrRNA基因序列X98418，因此后续科学家多依据这个序列对分离培养出的纳米细菌进行种属鉴定。目的：利用透射电镜，扫描电镜，茜素红染色和16SrRNA基因分析的方法对分离培养得到的纳米细菌进行进一步鉴定。方法：①透射电镜观察：纳米细菌14,000g高速离心沉淀，戊二醛前固定，四氧化二锇后固定，梯度脱水，环氧树脂包埋，制作超薄切片，双重电子染色，双蒸水洗涤，干燥后在透射电镜下观察拍照。②扫描电镜观察：纳米细菌沉淀利用戊二醛固定,常规脱水，，临界点干燥，喷金，在扫描电镜下观察拍照。③茜素红染色：培养标本涂片固定，冲洗，2％茜素红染液染，0.2％淡绿水溶液复染，O.5％醋酸水溶液冲洗,乙醇脱水，烘干后用中性树胶封片，高倍镜下观察拍照。④16SrRNA基因序列分析：提取纳米细菌基因组，设计纳米细菌16SrRNA基因序列特异性引物，PCR扩增其特异性序列，凝胶电泳观察并回收目的片段，送检进行基因序列分析，结果与GenBank比对。根据比对结果设计新发现纳米细菌16SrRNA基因特异性引物，对胎盘钙化组织进行16SrRNA基因序列分析，其结果与新发现纳米细菌进行比对，验证提取过程有无污染菌。再将所得菌株与GenBank中所收录纳米细菌相关16SrRNA基因做系统进化分析，绘制系统进化树。结果：①透射电镜下可见菌液中的纳米细菌呈椭圆形，直径在200-500nm，有或没有高电子密度的外壳包绕在外。②扫描电镜下可见纳米细菌形态微小，单个颗粒直径在200nm左右，或聚集成微米大小团块。③茜素红染色可见纳米细菌染成红色细小颗粒。④16SrRNA基因分析发现胎盘钙化组织中分离培养得到纳米细菌新种属，分别与纳米细菌X98418相比有93%和81%相似性，两个种属纳米细菌的16SrRNA基因测序结果提交GenBank得到基因编号JN029830与JF823648。钙化胎盘组织中纳米细菌基因扩增分析可知其与先前分离培养菌液中的纳米细菌是同一种菌。与GenBank中其他纳米细菌种属进行对比，发现这两个种属的纳米细菌与先期Kajander教授提交的Nanobacterium sanguineum(X98418)亲缘关系最近。结论：胎盘钙化组织中分离培养得到的纳米细菌是两个新的纳米细菌种属，与前期Kajander教授提交的Nanobacterium sanguineum具有较近的亲缘关系。背景：纳米细菌诱导钙化形成的机制还不明确，也没有相关实验研究证明它可以促进钙化相关蛋白的分泌。由于纳米细菌的主要成分是羟基磷灰石，而这种成分被证实可以促使骨骼形成同时促进钙化相关蛋白的分泌。因此，作者初步分析钙化相关蛋白--骨形态发生蛋白2（Bone morphogenetic protein BMP-2）与骨桥蛋白（Osteopontin OPN）在钙化胎盘组织中的分泌以及与纳米细菌感染之间的关系。目的：探讨纳米细菌感染对胎盘组织中骨形态发生蛋白2（BMP-2）与骨桥蛋白（OPN）两种钙化相关蛋白分泌的影响。方法：分别取钙化胎盘组织纳米细菌分离培养阳性组，阴性组以及正常胎盘组的胎盘组织标本作为实验对象。①免疫组化的方法分析不同实验组织BMP-2与OPN两种蛋白表达的差异：制作组织切片，HE染色，观察钙化。常规脱蜡水化，血清封闭，分别孵育BMP-2和OPN蛋白单克隆抗体4℃过夜，生物素化二抗作用，DAB显色，苏木素复染，显微镜下观察拍照。②Western Blot：SDS－PAGE电泳的方法分离出钙化与非钙化组织中BMP-2与OPN两种蛋白，化学发光，显影，定影，软件半定量分析目的条带的灰度值，比较BMP-2与OPN两种钙化相关蛋白在不同组织中的表达差异。结果：免疫组化结果可见钙化胎盘组织中纳米细菌分离培养阳性组与阴性组BMP-2与OPN的表达均高于正常胎盘组织(P0.01)。Western Blot结果分析可知BMP-2与OPN在钙化胎盘组织中纳米细菌分离阳性组和阴性组的表达高于正常胎盘组织(P0.01)。但两组之间表达差异不明显（P0.05）结论：钙化组织BMP-2与OPN两种蛋白的表达确有增加，但纳米细菌感染对于促进胎盘组织的钙化机制仍不明确。背景：纳米细菌的特殊组成成分、大小和复制方式使得其分离培养与保存方法一直没有统一。为了使更多疑似与纳米细菌感染有关的疾病可以分离培养出纳米细菌，作者以胎盘钙化组织为例，探讨钙化组织中纳米细菌的分离技术。同时，通过对纳米细菌不同的培养和保存条件的实验研究，来探讨纳米细菌的培养与保存的最佳条件和方法，有助于纳米细菌的阳性培养和后续基因组计划的实施。目的：以钙化的胎盘组织为例，寻求在钙化组织中分离纳米细菌的最佳方法，探究培养和保存纳米细菌最适宜的方法和条件。方法：方法①胎盘钙化组织标本分别用盐酸脱矿与超声振荡脱矿的方法分离纳米细菌，计算其分离阳性率。②分离出的纳米细菌分别用细胞培养箱与细菌培养箱培养4周，利用分光光度计记录两种条件下纳米细菌浓度的变化，并描绘生长曲线。③分别用4℃，-20℃与-80℃冰箱保存钙化组织和纳米细菌，记录纳米细菌分离和复苏的生长状况并绘制生长曲线。结果：①钙化组织用盐酸脱矿更易分离得到纳米细菌。②细胞与细菌培养环境下纳米细菌的生长速度并无明显差别。③4℃保存钙化组织和纳米细菌菌液对于其分离和复苏都要优于-20℃和-80℃ 结论：对钙化组织进行盐酸脱矿可以更好的分离出纳米细菌，并且可以在细菌培养箱内培养纳米细菌，新鲜钙化组织标本和纳米细菌可以短时间保存在4℃。
[Abstract]:Background: Professor Kajander and his team, Finland scientist, and his team first discovered cell vacuoles from mammalian cell culture, analyzed it in the absence of all possible microbial contamination, and found a microorganism that could be self replicating mineralization with a diameter of only 50-800nm, named nanoscale (Nanobacte RIA NB) and application for patent 1., the Fourier transform dispersive spectroscopy (FTIR) was used to show that the main component of the nanoscale shell is hydroxyapatite, so it is also called nano mineralized particle (CNPs).
Objective: to isolate and cultivate nanometallic bacteria related to calcification from placental calcification and fetal umbilical cord blood, to observe its growth characteristics, to explore the evidence of microorganism infection in placental calcification, and to investigate the relationship between placental calcification and fetal nanoscale bacterial infection.
Methods: 36 placental calcification tissues were taken as the research object, and the nanoscale in calcified tissue was observed by transmission electron microscope. Then, 1mol/L hydrochloric acid demineralization, Tris neutralization, saline dilution, 14000g high speed centrifugation were used. Finally, the method was separated through the filter of 0.22 m bacteria filter. Filter filtrate was filtered to the test tube with cotton plug, and the filter solution was added to the test tube with cotton plug. In the RPMI1640 cell culture medium containing 10% fetal bovine serum, the pH7.4 was adjusted, the cell culture box at 37 C and the 5%CO2,95% air were cultured. The growth of the bacteria was observed and the relative concentration of nanoscale bacterial liquid was recorded at the.OD650 wavelength of the precipitation type, and the growth curve was made. The fetal bovine serum (FBS) and the physiological saline were used as the reagents. The normal placental tissue without calcification was used as the experimental control, and the RPMI1640 medium was used as a blank control. At the same time, the umbilical cord blood of the fetus with placental calcification was taken from the umbilical cord blood, and the nanoscale bacteria were isolated and cultured.
Results: an elliptical placental tissue specimen was observed under transmission electron microscopy. There was an elliptical nano particle between placental villus and calcified plaque. The diameter of the nanoparticles was about 50-500nm. Each particle was surrounded by a thin shell with different electron densities. Some of the particles were visible and split. In the experimental group, 28 cases were available. The white precipitation was completely or partially adhered to the bottom of the test tube, and the control group had no precipitation. The difference was significant (P 0.01).OD650 spectrophotometer. The growth curve of nanoparticles was similar to that of other bacteria. The results of umbilical cord blood isolation and culture were positively related to the results of nanoscale culture in the corresponding calcified tissue. There were 12 cases. White sediment was observed in the culture samples, but not in the control group (P 0.01).
Conclusion: the nano particles with self replicating mineralization can be obtained from the calcified placenta by the method of isolation and culture of nanoscale, and the fetus of placental calcification is also easier to infect the nanoparticles.
Background: to further identify the morphology and genomics of the cultured white precipitates. Because the morphology of the nanometers is small and some are less than 100nm, many scientists believe that it does not exist and does not belong to Professor.Kajander, a biological category, to submit the specific 16SrRNA gene sequence X98 of nanoscale bacteria to GenBank. 418, therefore, subsequent scientists based on this sequence were used to identify species of nanomaterials isolated and cultured.
Objective: to further identify the nanomaterials isolated and cultured by transmission electron microscopy, scanning electron microscopy, alizarin red staining and 16SrRNA gene analysis.
Methods: (1) transmission electron microscopy: nanometrobacterium 14000g high speed centrifuge precipitation, glutaraldehyde fixed, four oxidation two osmium fixed, gradient dehydration, epoxy resin embedding, ultra-thin section, double electron staining, double water washing and observation under transmission electron microscope. 2. Scanning electron microscope observation: nanometroms precipitation using glutaraldehyde Fixed, dehydrated, critical point drying, spray gold, observation and photograph under scanning electron microscope. 3. Alizarin red staining: culture specimen smear fixed, rinse, 2% alizarin red dye dye, 0.2% light green water solution, O.5% acetic acid water solution rinse, ethanol dehydration, after drying with neutral gum seal, high times mirror observation taking pictures. (4) 16SrRNA gene sequence Analysis: the genome of nanoscale bacteria was extracted, the specific primers of the nanoscale 16SrRNA gene sequence were designed, the specific sequence was amplified by PCR, the target fragments were observed and recovered by gel electrophoresis, the gene sequence analysis was carried out and the results were compared with the GenBank. According to the comparison results, the specific primers of the nanoscale 16SrRNA gene were designed, and the placental calcium was found. 16SrRNA gene sequence analysis was carried out in the chemical tissue, and the results were compared with the new nanoscale bacteria. The results showed that there were pollution-free bacteria in the extraction process, and then the 16SrRNA gene related to nanbacteria in GenBank was analyzed, and the phylogenetic tree was plotted.
Results: under transmission electron microscopy, the nanometers in the bacteria were elliptical, the diameter of the bacteria was in 200-500nm, and the outer shell with or without high electron density was around. 2. Under the scanning electron microscope, the morphology of nanoscale was small, the diameter of the single particle was about 200nm, or the micromass mass was aggregated. 3. The alizarin red staining showed that the nanoscale bacteria were dyed red. 16SrRNA gene analysis found that the new species of nanoscale were isolated and cultured in placental calcification, and 93% and 81% were similar to nano bacteria X98418 respectively. The 16SrRNA gene sequencing results of two species of nanoscale were submitted to GenBank to obtain the nanoscale gene gene in JN029830 and JF823648. calcified placenta. The amplification analysis showed that the nanoscale bacteria in the previously isolated culture bacteria were the same bacteria. Compared with the other nanoscale species in GenBank, the nanoscale of the two species was found to be closest to the Nanobacterium sanguineum (X98418) submitted by Professor Kajander.
Conclusion: the isolation and culture of nanoscale in placental calcification is two new species of nanoscale, which is closely related to Nanobacterium sanguineum submitted by Professor Kajander in the earlier period.
Background: the mechanism of nanoscale induced calcification is not clear, and no related experimental studies have shown that it can promote the secretion of calcium related proteins. As the main component of the nanoscale is hydroxyapatite, this component has been proved to promote bone formation and promote the secretion of calcium related proteins. The relationship between calcium related protein - bone morphogenetic protein 2 (Bone morphogenetic protein BMP-2) and osteopontin (Osteopontin OPN) in calcified placental tissue and with nanoscale bacterial infection.
Objective: To investigate the effect of nanomaterials infection on the secretion of bone morphogenetic protein 2 (BMP-2) and osteopontin (OPN) two calcification associated proteins in placenta.
Methods: the positive group, the negative group and the placental tissue of the normal placenta group were taken as the experimental objects, respectively. (1) the difference of the expression of two proteins between BMP-2 and OPN in different experimental tissues was analyzed by immunohistochemistry: tissue section, HE staining, calcification, routine dewaxing, and serological seal Closed, incubating BMP-2 and OPN protein monoclonal antibodies at 4 C overnight, biotinylated two anti action, DAB coloration, hematoxylin redyeing, and microscopic observation under microscope. (2) Western Blot:SDS PAGE electrophoresis method was used to separate two kinds of protein of BMP-2 and OPN in calcified and non calcified tissues, chemiluminescence, development and fixing, and the software semi quantitative analysis of the target strip Compare the expression of two calcification related proteins between BMP-2 and OPN in different tissues.
Results: the results of immunohistochemistry showed that the expression of BMP-2 and OPN in the positive group and negative group in the calcified placenta tissue and negative group were higher than the normal placental tissue (P0.01).Western Blot results. It was found that the expression of BMP-2 and OPN in the positive and negative groups in the calcified placenta tissue was higher than that of the normal placental tissue (P0.01). However, the difference of expression between the two groups was not obvious (P0.05)
Conclusion: the expression of BMP-2 and OPN two proteins in calcified tissue is increased, but the mechanism of nanomaterials infection on promoting placenta calcification is still unclear.
Background: the special components, size and replication of nanoscale have not been unified. In order to isolate and culture more suspected diseases related to nanoscale bacterial infection, the authors take placental calcification as an example to explore the separation technology of nanoscale in calcified tissue. The best conditions and methods for the culture and preservation of nanoscale bacteria are discussed by the experimental study of the different culture and preservation conditions of nanoscale. It is helpful to the positive culture of nanoscale and the implementation of the follow-up genome project.
Objective: to seek the best method of separating nanoscale in calcified tissue by taking calcified placenta as an example, and to explore the most suitable methods and conditions for the cultivation and preservation of nanometers.
Methods: (1) the separation of nanbacteria by demineralization by hydrochloric acid and demineralization by ultrasonic oscillation was used to calculate the isolation positive rate. (2) the nanometers were cultured in cell culture box and bacterial culture box for 4 weeks, and the changes in the concentration of nanoscale under two conditions were recorded by spectrophotometer. The calcified tissue and nanoscale were stored at 4, -20 and -80, respectively. The growth conditions of nanoscale isolation and resuscitation were recorded and the growth curve was drawn.
Results: (1) there is no obvious difference in the growth rate of nanoscale by the demineralization of calcic tissue with hydrochloric acid. (2) the growth rate of nanoscale in cell and bacteria culture environment is not obviously different. (3) the preservation of calcified tissue and nanoscale bacterial liquid at 4 degrees is better than -20 C and -80 C for its separation and recovery.
Conclusion: the demineralization of hydrochloric acid in calcified tissue can better separate the nanoscale bacteria, and the nanoscale bacteria can be cultured in the bacterial culture box. The fresh calcified tissue specimen and the nanoscale can be kept at 4 C for a short time.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R378

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